Early expression of monomeric and oligomeric alpha-synuclein and tyrosine hydroxylase following intranigral injection of lipopolysaccharide

Early expression of monomeric and oligomeric alpha-synuclein and tyrosine hydroxylase following intranigral injection of lipopolysaccharide | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Early expression of monomeric and oligomeric alpha-synuclein and tyrosine hydroxylase following intranigral injection of lipopolysaccharide Alma Karen Lomeli-Lepe, Silvia Josefina López-Pérez, José Luis Castañeda-Cabral, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4253562/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The insoluble tangles of alpha-synuclein (α-syn) protein in the nigrostriatal circuit, characteristic of synucleinopathy, originate from low molecular weight oligomers, whose appearance and dissemination are related to neuroinflammation. These oligomeric forms of α-syn are considered highly cytotoxic but transient, so knowing the timing in which they appear remains a challenge. Therefore, this study aimed to analyze the abundance of oligomeric forms of α-syn and tyrosine hydroxylase (TH) between 1 and 7 days after inducing neuroinflammation with lipopolysaccharide ( LPS). Methods and Results LPS (2.5 µg/2.5 µL) was stereotaxically injected in the SN of adult male Wistar rats, which were sacrificed 3, 5 and 7 days after this intervention. The brains were processed for semi quantitative Western blot, along with brains from control and sham animals. Our results show an increased expression of α-syn monomer (15 kDa) only 3 days after LPS infusion, and the formation of 50 KDa and 60 kDa α-syn oligomers in the SN and STR between 3 and 7 days after LPS infusion. Furthermore, the presence of these oligomers was accompanied by a decrease in the expression of nigral TH. Conclusion These findings highlight the rapidity with which potentially toxic forms of α-syn appear in the nigrostriatal circuit after a neuroinflammatory challenge, in addition to allowing us to identify specific oligomers and a temporal relation with neurodegeneration of TH-positive cells. Knowledge of the timing and location in which these small oligomers appear is essential to developing therapeutic strategies to prevent its formation. α-syn oligomers nigrostriatal circuit synucleinopathies tyrosine hydroxylase Figures Figure 1 Figure 2 Figure 3 1. Introduction Synucleinopathies constitute a group of neurodegenerative diseases that share a major pathological hallmark, the misfolding and aggregation of the alpha-synuclein protein (α-syn). This aggregation is a complex process involving several stages, from monomeric α-syn in its native form (15 kDa) to various, dynamic oligomeric forms, ultimately evolving into a fibrillar form [ 1 – 3 ]. Although there is no specific known cause that promotes α-syn aggregation, factors such as exposure to environmental toxins, pesticides, and other neurotoxins [ 3 – 5 ] can influence this process, leading to downstream cytotoxic events that result in neuronal cell death. The different cellular locations and patterns of α-syn deposition give rise to clinically distinct entities among the synucleinopathies, including pure autonomic failure, multiple system atrophy, dementia with Lewy bodies, and Parkinson's disease (PD) [ 6 ], with PD being the most extensively characterized. Tyrosine hydroxylase (TH) positive dopaminergic neurons located in the substantia nigra pars compacta (SNpc) are particularly vulnerable to degeneration due to the accumulation of oligomeric and fibrillar forms of α-syn [ 7 ]. These neurons send inputs to the striatum (STR), forming the nigrostriatal circuit. Owing to the structure of this circuit, the gradual degeneration of nigral neurons during disease development leads to a reduction in dopamine in the STR, causing the characteristic motor deficits observed in PD [ 8 ]. While the origins of these conditions remain incompletely understood, years of accumulated evidence point to two fundamental and relatively common processes during the initial phases of these neurological disorders: neuroinflammation and oxidative stress. Several neurotoxic molecules are employed to create experimental animal models that exhibit both of these characteristics at the onset and progression phases of the disease [ 9 ]. However, in the context of dopaminergic nigral neuron degeneration linked to PD, it has proven challenging to ascertain whether they are a cause or a consequence of other processes involved in the development of the condition [ 10 ]. Lipopolysaccharide (LPS), a bacterial endotoxin with the ability to activate glial cells [ 11 , 12 ], also induces α-syn aggregation in rodents [ 13 ]. Consequently, LPS has been utilized to investigate the involvement of different cytotoxic effectors in α-syn aggregation and the dopaminergic neurodegeneration associated with PD. Although the mechanism inducing the overexpression of α-syn and the transformation of native α-syn to higher-order aggregates is still unknown, it is known that once this process occurs, the heightened presence of α-syn maintains a continual state of glial activation and excessive reactive oxygen species (ROS) production in a continuous feedback process [ 14 – 17 ]. This process amplifies the neuroinflammatory and oxidative feedback loop [ 18 ]. However, the notion that the initial stages of α-syn aggregation are the most toxic, at least for nigral dopaminergic neurons, has been gaining traction in recent years. Determining the exact size of these early forms of α-syn is challenging, as they likely emerge very early in the disease process and persist for only a brief time. Nevertheless, characterizing these oligomers is crucial due to its high toxicity. With this aim in mind, we structured this study to evaluate the presence of the native form of α-syn (15 kDa) and the closest oligomeric forms (37, 50 and 60 kDa) in the SN and STR. Additionally, we analyzed the survival of nigral dopaminergic cells in relation to the presence of these oligomeric forms of α-syn. 2. Materials and methods 2.1 Ethical considerations, experimental design, and treatments The experiments carried out in this study received the approval of the local Bioethics through the Research Coordination of the University Campus of Biological and Agricultural Science (CUCBA) of the University of Guadalajara, under the agreement CINV.104/12. The experiments were aligned with the Mexican Official Norms (NOM-062-ZOO-1999 and NOM-033-ZOO-1995) and Directive 2010/63/EU. Also, in the conduct and reporting of our study, we adhered to the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) 2.0. Every effort was made to minimize the number of animals and the distress experienced by the animals during the experiment. Thirty-five adult male Wistar rats weighing between 200 and 250 g each were used in this study. The animals were housed within a CUCBA vivarium, two per cage, under a 12:12 h light:dark cycle and ad libitum access to food and water. To start the experimental process, the animals were randomly assigned to the following groups: the control group (which was not treated), the sham group (which received only the vehicle: 0.9% saline solution), or the LPS group (which received LPS from Escherichia coli , O111:B4, Sigma‒Aldrich, USA) at a dosage of 2.5 µg/2.5 µl, diluted in vehicle. This solution was kept at 4°C and protected from light during the entire procedure. Each group consisted of 5 animals. On day 1, rats in the sham and LPS groups were anesthetized with 4% isoflurane in a ventilated anesthesia chamber and mounted in a stereotaxic frame with the head immobilized using ear and incisor bars. A small hole was created in the skull at the level of the SN in the right hemisphere, following the coordinates of Paxinos and Watson [ 19 ]: AP -5.8 mm, L -1.0 mm, V -7.8 mm. Vehicle or LPS was delivered into the SN using a 50 µL microsyringe (Hamilton Company, Nevada, USA) connected to a microinfusion pump (Fusion 200, Chemyx Inc., USA) at a controlled flow rate of 1 µL/min for 2.5 min. Then, the needle was held in place for 3 min before being slowly retracted. Once this procedure was completed, the hole was sealed using cyanoacrylate, and the skin incision was sutured, cleaned and disinfected. Once the animals recovered from anesthesia, they were returned to their respective cages. 2.2 Measurement of α-syn and tyrosine hydroxylase expression by Western blot To assess early α-syn expression in response to intranigral LPS injection, animals were euthanized at 3, 5, and 7 days post injury (DPI) by an intraperitoneal overdose of sodium pentobarbital (60 mg/kg). The brains were rapidly removed and then placed on an ice-cold plate to be dissected region by region. Ipsilateral and contralateral SN and STR tissues were dissected, weighed, and subsequently frozen at -20°C. Afterward, these tissue samples were thawed but kept at 4°C, homogenized by sonication in lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 20 mM NaF, 0.5 mM Na 3 VO 4 , and 1% Tergitol type NP-40) and supplemented with a protease inhibitor cocktail, according to manufacturer's instructions ( cOmplete™ Protease Inhibitor Cocktail , cat. 04693116001, Roche, Germany). Homogenates were centrifuged at 13,000× g for 30 min at 4°C, in a Sorvall Legend Micro 21R centrifuge (Thermo Scientific, Germany). The supernatant was collected, aliquoted, and frozen at -20°C until protein analysis. The protein concentration was determined according to the Lowry method [ 20 ] with a DC Protein Assay Kit (cat. 5000116; Bio-Rad Laboratories, USA) on a Multiskan Go spectrophotometer (Thermo Scientific, Finland), using bovine serum albumin (cat. 500-0007, Bio-Rad Laboratories, USA) as an external standard. Briefly: twenty micrograms of total protein from each supernatant was denatured in 5 µL of Laemmli buffer (500 mM Tris-HCl pH 6.8, 2% sodium dodecyl sulfate (SDS), 10% glycerol, 10% β-mercaptoethanol, and 0.1% bromophenol blue) at 95°C for 5 min. These samples were then loaded on a 12% SDS-polyacrylamide (SDS‒PAGE) gel for electrophoretic separation. Electrophoresis was conducted at 85 V for 30 min and 95 V for 2 h to separate the proteins. These proteins were subsequently transferred onto a nitrocellulose membrane (Protean Premium 0.45 µm, Amersham, Germany), at 110 V for 30 min. Afterwards, the membranes were blocked in a 5% blocker solution (QuickBlocker, EMD Millipore, USA) dissolved in 0.1 M PBS with 0.1% Tween-20 at 4°C for 1 h. The membrane was then immunolabeled using commercially available antibodies against monomeric and oligomeric α-syn (1:1000-1:2000; Cat. ab212184, Abcam, Cambridge, UK), and TH (1:1000; Cat. ab112, Abcam, Cambridge, UK). The membranes were incubated with these primary antibodies at 4°C overnight and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000; Cat. 926–8000, LI-COR Bioscience, USA) at 4°C for 2 h. Subsequently, the membranes were exposed to chemiluminescent substrate (SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Scientific, USA). The signals corresponding to the proteins of interest were acquired using a C-DiGit Blot Scanner (LI-COR Bioscience, USA), and the bands were analyzed using Image Studio Lite 3.1.4 software (LI-COR Bioscience, USA). To normalize the expression of monomeric α-syn and TH, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was employed as a loading control (1:5000; Cat. ab125247, Abcam, Cambridge, UK), while the oligomeric forms of α-syn (35–60 kDa) were normalized to monomeric α-syn (15 kDa). All samples were analyzed in duplicate. 2.3 Statistical analysis The data are expressed as the mean ± SD for each measured parameter. For data analysis, one-way ANOVA with Tukey's post hoc test was applied. Statistical significance was considered when p < 0.1. Statistical analyses and graphical representations were performed using GraphPad Prism v.8 software. 3. Results 3.1 Expression of monomeric and oligomeric α-syn following LPS injection in the SN At 7 DPI, the abundance of the monomeric α-syn form (15 kDa) in the SN ipsilateral to the lesion exhibited a slight increase in the LPS group in comparison to the control and sham groups (Fig. 1a). Within the same samples, we identified bands corresponding to oligomeric forms of α-syn at 37, 50, and 60 kDa (Fig. 1e). Notably, the 50 kDa oligomeric form showed an increase on the contralateral side at 5 DPI compared to the control group (Fig. 1c), and the 60 kDa oligomeric form displayed an increase ipsilateral to the lesion at 5 DPI compared to the control group (Fig. 1d). However, no significant changes were observed in the 37 kDa oligomers (Fig. 1b). These findings confirm the efficacy of LPS in inducing α-syn expression, as well as the rapid emergence of various oligomeric forms in the SN during the early stages following the insult. 3.2 Increased expression of monomeric and oligomeric α-syn in the STR following nigral LPS injection The STR is also affected in synucleinopathies due to its direct connections with the SN. To understand the timeline of oligomer formation in this region, we conducted a similar analysis at 3, 5, and 7 DPI within the STR. The results revealed an early increase in the expression of monomeric α-syn (15 kDa) at 5 DPI on both ipsilateral and contralateral sides of the lesion in comparison to the control group (Fig. 2 a). However, no changes were observed in the 37 kDa (Fig. 2 b) or 50 kDa oligomers in this region (Fig. 2 c), while the 60 kDa form was increased at 5 and 7 DPI only on the ipsilateral side to the LPS injection, compared with the control group (Fig. 2 d). Interestingly, we also identified an increased abundance of the 60 kDa form on the contralateral side at 7 DPI in the LPS group compared to the control group (Fig. 2 d). These results suggest a swift dissemination of oligomers from the SN to the STR, in response to LPS stimulation. 3.3 TH expression in the SN and STR The degeneration of dopaminergic neurons in the SN stands as a defining characteristic of synucleinopathies. To assess the impact of modified α-syn expression prompted by LPS stimulation on the survival of TH positive cells and terminals, the expression of TH was evaluated at 3, 5, and 7 DPI in SN as well as the STR. In line with the amplified α-syn expression in the SN at 7 DPI, there was a concurrent decrease in TH expression on the side ipsilateral to the LPS injection at 5 and 7 DPI compared with the control group (Fig. 3 a). Within the STR, a reduction in TH expression compared to the control group was observed solely on the side ipsilateral to the injection, and solely at 3 DPI, with no discernible changes at 5 or 7 DPI, or comparing with the contralateral side (Fig. 3 c). Together, these results suggest a rapid degeneration of TH positive (presumably dopaminergic) cells and terminals as a consequence of the reaction triggered by LPS. 4. Discussion The inflammatory process and the consequent aggregation of α-syn are relevant in the development and progression of synucleinopathies; these events can be recapitulated by using intracerebral LPS, which acts via microglial activation and secretion of neurotoxins, generating an inflammatory microenvironment [ 21 , 22 ]. In this experiment, we employed intracerebral LPS infusion with the aim of analyzing the expression and early dynamics of α-syn, in the interconnected cerebral nuclei SN and STR once a neuroinflammatory process is triggered, and the influence on the dopaminergic cells and terminals. Our findings indicate that the inflammatory reaction induced by LPS impacts the expression levels of α-syn and TH in the SN and STR. Additionally, we observed a rapid emergence of small oligomers, ranging from 15 to 60 kDa, exhibiting particular patterns of appearance and expression in the SN and STR. This indicates a cause-and-effect relationship between LPS-induced inflammation and α-syn expression. The general characteristics of the inflammatory reaction following intracerebral LPS infusion have been previously described in murine models [ 23 ], along with its ability to increase the accumulation and aggregation of α-syn in the SN [ 24 – 26 ]. In contrast to other drugs, such as 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produce a similar outcome within a few hours of damage, the neurodegenerative process triggered by LPS is relatively delayed, since occurs more slowly, resembling the slow progression of synucleinopathy [ 27 – 29 ], which allows analyzing the presence and abundance of early oligomeric forms of α-syn. We observed an increase in the 15 kDa form of α-syn in the STR as early as the third day after LPS administration, but not in the SN. This could indicate the presence of monomers before 3 DPI in the SN, maybe in a physiological abundance sufficient to maintain cellular functions of α-syn, but not enough to be detected by Western blot. In fact, these 15 kDa monomeric forms were detected in the SN up to 7 DPI, supporting the notion of an indetectable quantity of them, and a posterior accumulation in the SN. Furthermore, it is known that the expression of this protein is generally higher in the STR than in the SN, probably due to its affinity for small-sized vesicles, such as presynaptic vesicles [ 39 ]. To our knowledge, this is the first report of abnormally elevated levels of monomeric α-syn under these conditions as early as 3 days after LPS injection in the nigrostriatal circuit. Nonetheless, the level of monomer expression that we observed in the STR at 3 DPI and in the SN at 7 DPI are not a normal state. It has been proposed that the pathogenicity of α-syn is related to the formation of abnormal aggregates, which causes it to lose its normal function, or to gain functions that it did not have before [ 40 ]. Notably, there are no reports of the disruption of cellular functions attributed to α-syn, such as neurotransmitter release or mitochondrial functions [ 41 ] under conditions of induced inflammation and neurodegeneration. It will be necessary to delve deeper into this topic, to recognize the perhaps subtle pathological manifestations that we are not showing, in these early stages of α-syn aggregation. Interestingly, we observed an increase in the expression level of the 60 kDa oligomer (potentially corresponding to a tetrameric α-syn) at 5 DPI in both the SN and the STR. This finding reinforces our hypothesis concerning the instability of the monomer and its transformation to more stable forms, such as the tetramer, in the SN. In addition, it sheds light on the rapid dissemination of these aggregated forms toward the striatum. Recently, it was suggested that the tetrameric form tends to resist α-syn aggregation as long as it remains with an α-helical configuration. [ 30 ]. However, if these tetramers shift into β-sheets, they can indeed promote α-syn aggregation [ 31 ]. It is also plausible that α-helix and β-sheet configurations coexist during the initial stages of LPS-induced damage, and depending on the permanency and amplification of the inflammatory process, the balance tips towards self-regulation, or towards dissemination and neurodegeneration. Further research will be imperative to ascertain whether the presence of the tetramer in the α-helix form truly provides neuroprotection and stability. A parallel study noted a significant increase in trimeric α-syn (51 kDa) expression in the SN seven days after LPS stimulation, although other oligomers were not observed [ 32 ]. While we did not detect significant changes in abundance of 37 or 50 kDa oligomers, another study utilizing intranigral LPS in adult rats identified an increased presence of the 50 kDa form seven days after LPS infusion [ 32 ]. In our study, we administered LPS at a lower concentration (2.5 µg/µL) than Liu et al. (2020) [ 32 ], trying to mimic a slow onset, as is believed to happen in human synucleinopathy. Although we did not observe a significant increase in 50 kDa oligomers, we obtained evidence of a rise in its expression. Although this increase did not reach the threshold of statistical significance set forth by the study, its presence is indicative of potential neurotoxicity and we consider that it is evidence that the aggregation process is happening [ 32 – 34 ]. Finally, we observed a decrease in TH levels at 5 DPI, associated with the presence of various α-syn oligomeric species generated in the SN after LPS infusion. Previous evidence has indicated that α-syn overexpression leads to reduced TH content in the SN [ 35 , 36 ], with the involvement of Ser40, whose phosphorylation significantly contributes to TH activation and dopamine synthesis [ 37 , 38 ]. This result supports the hypothesis that high levels of α-syn expression are toxic to TH-positive dopaminergic neurons, although our approach does not allow us to determine if there is neurodegeneration or if the detrimental effect is on the expression of the protein. Few studies have examined the time course of both monomeric and oligomeric α-syn levels within a short timeframe. Our work is the first to detect these aggregates early in an in vivo model. In other studies, accumulation of α-syn has been observed in the cytoplasm of spared TH-positive neurons four weeks after intrastriatal or intrapallidal LPS administration [ 24 ],25]. In another study, intraperitoneal injection of LPS in aged C57BL/6J mice resulted in an increased abundance of monomeric α-syn one day after LPS administration, although no data were presented regarding oligomeric forms greater than 15 kDa [ 26 ]. Despite differences in the way neuroinflammation is induced, LPS dosage, species, age, and administration method, this result underscores the rapid response of α-syn to brain inflammation, in this case triggered by LPS. 5. Conclusions Our findings conclusively establish that the injection of LPS into the SN swiftly influences α-syn expression, resulting in the formation of oligomers. This finding aligns with the overarching hypothesis that inflammatory processes play a pivotal role in synucleinopathies. Furthermore, the emergence of these oligomeric forms, which are being shown for the first time in this in vivo animal model, underscores the vulnerability of nigral dopaminergic neurons and their interconnectedness with the STR. This vulnerability becomes evident when potentially toxic forms of α-syn form rapidly following a minimal dose of LPS, impacting both the SN and the STR in a few days, placing a seed that can compromise future brain functioning. Enhancing our comprehension of the cellular and molecular mechanisms driving the onset of α-synucleinopathies will undoubtedly contribute to the future development of therapeutic strategies aimed at mitigating the progression of this pathology. Declarations Ethical approval was taken from the Research Coordination of the University Campus of Biological and Agricultural Science (CUCBA) of the University of Guadalajara, under the agreement CINV.104/12. All authors reviewed and authorized the final manuscript and agreed to publish it. AKLL did the experimental work and wrote the first draft under the supervision of SJLP. JLCC helped in data collection and data analysis. SJLP and MEUG critically review the final manuscript. All authors read and approved the final manuscript. The authors declare no competing interest. This work received funding from the Consejo Nacional de Humanidades, Ciencia y Tecnología (CONAHCYT) through scholarship No. 1028543 to AKLL., and from the University of Guadalajara through the P3E-UDG-2022/2023 program to Author Contribution AKLL did the experimental work and wrote the first draft under the supervision of SJLP. JLCC helped in data collection and data analysis. SJLP and MEUG critically review the final manuscript. All authors read and approved the final manuscript Data Availability Data is provided within the manuscript. Other data are available from the corresponding author upon reasonable request. References Mehra S, Sahay S, Maji SK (Oct. 2019) α-Synuclein misfolding and aggregation: Implications in Parkinson’s disease pathogenesis. Biochim Biophys Acta Proteins Proteom 1867(10):890–908. 10.1016/j.bbapap.2019.03.001 Koga S, Sekiya H, Kondru N, Ross OA, Dickson DW (Dec. 2021) Neuropathology and molecular diagnosis of Synucleinopathies. 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Nov. 2019. doi: 10.1155/2019/4246350 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4253562","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":290322532,"identity":"1ac3ed23-0352-4497-bbc6-4f0f1189e645","order_by":0,"name":"Alma Karen Lomeli-Lepe","email":"","orcid":"","institution":"Universidad de Guadalajara","correspondingAuthor":false,"prefix":"","firstName":"Alma","middleName":"Karen","lastName":"Lomeli-Lepe","suffix":""},{"id":290322535,"identity":"79c10370-40d4-44e8-ab9c-c3d920b3824f","order_by":1,"name":"Silvia Josefina López-Pérez","email":"data:image/png;base64,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","orcid":"","institution":"Universidad de Guadalajara","correspondingAuthor":true,"prefix":"","firstName":"Silvia","middleName":"Josefina","lastName":"López-Pérez","suffix":""},{"id":290322538,"identity":"43836278-de39-4a31-b125-f9d5c4196c8d","order_by":2,"name":"José Luis Castañeda-Cabral","email":"","orcid":"","institution":"Universidad de Guadalajara","correspondingAuthor":false,"prefix":"","firstName":"José","middleName":"Luis","lastName":"Castañeda-Cabral","suffix":""},{"id":290322541,"identity":"95983b09-cc19-4609-9c21-2fa0264ef985","order_by":3,"name":"Mónica E. Ureña-Guerrero","email":"","orcid":"","institution":"Universidad de Guadalajara","correspondingAuthor":false,"prefix":"","firstName":"Mónica","middleName":"E.","lastName":"Ureña-Guerrero","suffix":""}],"badges":[],"createdAt":"2024-04-11 16:20:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4253562/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4253562/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54758472,"identity":"42b19bbf-0b2c-45fd-b4ed-b013f8db9a65","added_by":"auto","created_at":"2024-04-16 10:37:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":426229,"visible":true,"origin":"","legend":"\u003cp\u003eSemiquantitative analysis of α-syn in the SN was performed using western blotting. (a-d) The expression of α-syn in monomeric (15 kDa) and oligomeric forms (37, 50, and 60 kDa) respectively. (e) Representative western blot images of α-syn monomers (15 kDa) and oligomers (37, 50, and 60 kDa), with GAPDH as the loading control. The data are presented as the mean ± SD. Data were analyzed by one-way ANOVA with a \u003cem\u003epost hoc\u003c/em\u003e Tukey test. *p\u0026lt;0.1 compared to the intact and sham groups (n = 5 per group).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4253562/v1/068e9b3649eaa82775f79203.png"},{"id":54758473,"identity":"0a7277a8-1755-42ac-aeef-37b465ede892","added_by":"auto","created_at":"2024-04-16 10:37:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":430474,"visible":true,"origin":"","legend":"\u003cp\u003eSemiquantitative analysis of α-syn in the STR was performed using western blotting. (a-d) The abundance of α-syn in monomeric (15 kDa) and oligomeric forms (37, 50, and 60 kDa). (e) Representative western blot images of α-syn monomers (15 kDa) and oligomers (37, 50, and 60 kDa), along with the loading control GAPDH. The data are presented as the mean ± SD. Data were analyzed by one-way ANOVA with a \u003cem\u003epost hoc\u003c/em\u003e Tukey test. *p\u0026lt;0.1 compared to the intact and sham groups (n = 5 per group).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4253562/v1/8b7029e649e1d16c073e68ac.png"},{"id":54758475,"identity":"0b3d1bb8-84c1-4c4e-9397-80919283a3e0","added_by":"auto","created_at":"2024-04-16 10:37:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":354247,"visible":true,"origin":"","legend":"\u003cp\u003eSemiquantitative analysis of TH in the SN and STR was conducted using western blotting analysis. (a, c) The expression of TH on the ipsilateral and contralateral sides. (b, d) Representative western blot images of TH and the loading control GAPDH. The data are presented as the mean ± SD. Data were analyzed by one-way ANOVA with a \u003cem\u003epost hoc\u003c/em\u003e Tukey test. *p\u0026lt;0.1 compared to the intact control and sham groups (n = 5 per group).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4253562/v1/3d55da73dc42005e7d670054.png"},{"id":54759587,"identity":"f929742d-663d-4320-a40f-15c929a91cef","added_by":"auto","created_at":"2024-04-16 10:53:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1156948,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4253562/v1/836f0930-52db-4454-a1e0-a06efec6be41.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Early expression of monomeric and oligomeric alpha-synuclein and tyrosine hydroxylase following intranigral injection of lipopolysaccharide","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSynucleinopathies constitute a group of neurodegenerative diseases that share a major pathological hallmark, the misfolding and aggregation of the alpha-synuclein protein (α-syn). This aggregation is a complex process involving several stages, from monomeric α-syn in its native form (15 kDa) to various, dynamic oligomeric forms, ultimately evolving into a fibrillar form [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Although there is no specific known cause that promotes α-syn aggregation, factors such as exposure to environmental toxins, pesticides, and other neurotoxins [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] can influence this process, leading to downstream cytotoxic events that result in neuronal cell death. The different cellular locations and patterns of α-syn deposition give rise to clinically distinct entities among the synucleinopathies, including pure autonomic failure, multiple system atrophy, dementia with Lewy bodies, and Parkinson's disease (PD) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], with PD being the most extensively characterized.\u003c/p\u003e \u003cp\u003eTyrosine hydroxylase (TH) positive dopaminergic neurons located in the substantia nigra pars compacta (SNpc) are particularly vulnerable to degeneration due to the accumulation of oligomeric and fibrillar forms of α-syn [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These neurons send inputs to the striatum (STR), forming the nigrostriatal circuit. Owing to the structure of this circuit, the gradual degeneration of nigral neurons during disease development leads to a reduction in dopamine in the STR, causing the characteristic motor deficits observed in PD [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhile the origins of these conditions remain incompletely understood, years of accumulated evidence point to two fundamental and relatively common processes during the initial phases of these neurological disorders: neuroinflammation and oxidative stress. Several neurotoxic molecules are employed to create experimental animal models that exhibit both of these characteristics at the onset and progression phases of the disease [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, in the context of dopaminergic nigral neuron degeneration linked to PD, it has proven challenging to ascertain whether they are a cause or a consequence of other processes involved in the development of the condition [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLipopolysaccharide (LPS), a bacterial endotoxin with the ability to activate glial cells [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], also induces α-syn aggregation in rodents [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Consequently, LPS has been utilized to investigate the involvement of different cytotoxic effectors in α-syn aggregation and the dopaminergic neurodegeneration associated with PD.\u003c/p\u003e \u003cp\u003eAlthough the mechanism inducing the overexpression of α-syn and the transformation of native α-syn to higher-order aggregates is still unknown, it is known that once this process occurs, the heightened presence of α-syn maintains a continual state of glial activation and excessive reactive oxygen species (ROS) production in a continuous feedback process [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This process amplifies the neuroinflammatory and oxidative feedback loop [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, the notion that the initial stages of α-syn aggregation are the most toxic, at least for nigral dopaminergic neurons, has been gaining traction in recent years.\u003c/p\u003e \u003cp\u003eDetermining the exact size of these early forms of α-syn is challenging, as they likely emerge very early in the disease process and persist for only a brief time. Nevertheless, characterizing these oligomers is crucial due to its high toxicity. With this aim in mind, we structured this study to evaluate the presence of the native form of α-syn (15 kDa) and the closest oligomeric forms (37, 50 and 60 kDa) in the SN and STR. Additionally, we analyzed the survival of nigral dopaminergic cells in relation to the presence of these oligomeric forms of α-syn.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Ethical considerations, experimental design, and treatments\u003c/h2\u003e \u003cp\u003e The experiments carried out in this study received the approval of the local Bioethics through the Research Coordination of the University Campus of Biological and Agricultural Science (CUCBA) of the University of Guadalajara, under the agreement CINV.104/12. The experiments were aligned with the Mexican Official Norms (NOM-062-ZOO-1999 and NOM-033-ZOO-1995) and Directive 2010/63/EU. Also, in the conduct and reporting of our study, we adhered to the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) 2.0. Every effort was made to minimize the number of animals and the distress experienced by the animals during the experiment.\u003c/p\u003e \u003cp\u003eThirty-five adult male Wistar rats weighing between 200 and 250 g each were used in this study. The animals were housed within a CUCBA vivarium, two per cage, under a 12:12 h light:dark cycle and \u003cem\u003ead libitum\u003c/em\u003e access to food and water. To start the experimental process, the animals were randomly assigned to the following groups: the control group (which was not treated), the sham group (which received only the vehicle: 0.9% saline solution), or the LPS group (which received LPS from \u003cem\u003eEscherichia coli\u003c/em\u003e, O111:B4, Sigma‒Aldrich, USA) at a dosage of 2.5 \u0026micro;g/2.5 \u0026micro;l, diluted in vehicle. This solution was kept at 4\u0026deg;C and protected from light during the entire procedure. Each group consisted of 5 animals.\u003c/p\u003e \u003cp\u003eOn day 1, rats in the sham and LPS groups were anesthetized with 4% isoflurane in a ventilated anesthesia chamber and mounted in a stereotaxic frame with the head immobilized using ear and incisor bars. A small hole was created in the skull at the level of the SN in the right hemisphere, following the coordinates of Paxinos and Watson [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]: AP -5.8 mm, L -1.0 mm, V -7.8 mm. Vehicle or LPS was delivered into the SN using a 50 \u0026micro;L microsyringe (Hamilton Company, Nevada, USA) connected to a microinfusion pump (Fusion 200, Chemyx Inc., USA) at a controlled flow rate of 1 \u0026micro;L/min for 2.5 min. Then, the needle was held in place for 3 min before being slowly retracted. Once this procedure was completed, the hole was sealed using cyanoacrylate, and the skin incision was sutured, cleaned and disinfected. Once the animals recovered from anesthesia, they were returned to their respective cages.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Measurement of α-syn and tyrosine hydroxylase expression by Western blot\u003c/h2\u003e \u003cp\u003eTo assess early α-syn expression in response to intranigral LPS injection, animals were euthanized at 3, 5, and 7 days post injury (DPI) by an intraperitoneal overdose of sodium pentobarbital (60 mg/kg). The brains were rapidly removed and then placed on an ice-cold plate to be dissected region by region. Ipsilateral and contralateral SN and STR tissues were dissected, weighed, and subsequently frozen at -20\u0026deg;C. Afterward, these tissue samples were thawed but kept at 4\u0026deg;C, homogenized by sonication in lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 20 mM NaF, 0.5 mM Na\u003csub\u003e3\u003c/sub\u003eVO\u003csub\u003e4\u003c/sub\u003e, and 1% Tergitol type NP-40) and supplemented with a protease inhibitor cocktail, according to manufacturer's instructions (\u003cem\u003ecOmplete\u0026trade; Protease Inhibitor Cocktail\u003c/em\u003e, cat. 04693116001, Roche, Germany). Homogenates were centrifuged at 13,000\u0026times;\u003cem\u003eg\u003c/em\u003e for 30 min at 4\u0026deg;C, in a Sorvall Legend Micro 21R centrifuge (Thermo Scientific, Germany). The supernatant was collected, aliquoted, and frozen at -20\u0026deg;C until protein analysis.\u003c/p\u003e \u003cp\u003eThe protein concentration was determined according to the Lowry method [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] with a DC Protein Assay Kit (cat. 5000116; Bio-Rad Laboratories, USA) on a Multiskan Go spectrophotometer (Thermo Scientific, Finland), using bovine serum albumin (cat. 500-0007, Bio-Rad Laboratories, USA) as an external standard. Briefly: twenty micrograms of total protein from each supernatant was denatured in 5 \u0026micro;L of Laemmli buffer (500 mM Tris-HCl pH 6.8, 2% sodium dodecyl sulfate (SDS), 10% glycerol, 10% β-mercaptoethanol, and 0.1% bromophenol blue) at 95\u0026deg;C for 5 min. These samples were then loaded on a 12% SDS-polyacrylamide (SDS‒PAGE) gel for electrophoretic separation. Electrophoresis was conducted at 85 V for 30 min and 95 V for 2 h to separate the proteins. These proteins were subsequently transferred onto a nitrocellulose membrane (Protean Premium 0.45 \u0026micro;m, Amersham, Germany), at 110 V for 30 min. Afterwards, the membranes were blocked in a 5% blocker solution (QuickBlocker, EMD Millipore, USA) dissolved in 0.1 M PBS with 0.1% Tween-20 at 4\u0026deg;C for 1 h. The membrane was then immunolabeled using commercially available antibodies against monomeric and oligomeric α-syn (1:1000-1:2000; Cat. ab212184, Abcam, Cambridge, UK), and TH (1:1000; Cat. ab112, Abcam, Cambridge, UK). The membranes were incubated with these primary antibodies at 4\u0026deg;C overnight and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000; Cat. 926\u0026ndash;8000, LI-COR Bioscience, USA) at 4\u0026deg;C for 2 h. Subsequently, the membranes were exposed to chemiluminescent substrate (SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Scientific, USA). The signals corresponding to the proteins of interest were acquired using a C-DiGit Blot Scanner (LI-COR Bioscience, USA), and the bands were analyzed using Image Studio Lite 3.1.4 software (LI-COR Bioscience, USA).\u003c/p\u003e \u003cp\u003eTo normalize the expression of monomeric α-syn and TH, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was employed as a loading control (1:5000; Cat. ab125247, Abcam, Cambridge, UK), while the oligomeric forms of α-syn (35\u0026ndash;60 kDa) were normalized to monomeric α-syn (15 kDa). All samples were analyzed in duplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD for each measured parameter. For data analysis, one-way ANOVA with Tukey's \u003cem\u003epost hoc\u003c/em\u003e test was applied. Statistical significance was considered when \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.1. Statistical analyses and graphical representations were performed using GraphPad Prism v.8 software.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Expression of monomeric and oligomeric α-syn following LPS injection in the SN\u003c/h2\u003e \u003cp\u003eAt 7 DPI, the abundance of the monomeric α-syn form (15 kDa) in the SN ipsilateral to the lesion exhibited a slight increase in the LPS group in comparison to the control and sham groups (Fig.\u0026nbsp;1a). Within the same samples, we identified bands corresponding to oligomeric forms of α-syn at 37, 50, and 60 kDa (Fig.\u0026nbsp;1e). Notably, the 50 kDa oligomeric form showed an increase on the contralateral side at 5 DPI compared to the control group (Fig.\u0026nbsp;1c), and the 60 kDa oligomeric form displayed an increase ipsilateral to the lesion at 5 DPI compared to the control group (Fig.\u0026nbsp;1d). However, no significant changes were observed in the 37 kDa oligomers (Fig.\u0026nbsp;1b). These findings confirm the efficacy of LPS in inducing α-syn expression, as well as the rapid emergence of various oligomeric forms in the SN during the early stages following the insult.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Increased expression of monomeric and oligomeric α-syn in the STR following nigral LPS injection\u003c/h2\u003e \u003cp\u003eThe STR is also affected in synucleinopathies due to its direct connections with the SN. To understand the timeline of oligomer formation in this region, we conducted a similar analysis at 3, 5, and 7 DPI within the STR. The results revealed an early increase in the expression of monomeric α-syn (15 kDa) at 5 DPI on both ipsilateral and contralateral sides of the lesion in comparison to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). However, no changes were observed in the 37 kDa (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) or 50 kDa oligomers in this region (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), while the 60 kDa form was increased at 5 and 7 DPI only on the ipsilateral side to the LPS injection, compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Interestingly, we also identified an increased abundance of the 60 kDa form on the contralateral side at 7 DPI in the LPS group compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). These results suggest a swift dissemination of oligomers from the SN to the STR, in response to LPS stimulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.3 TH expression in the SN and STR\u003c/h2\u003e \u003cp\u003eThe degeneration of dopaminergic neurons in the SN stands as a defining characteristic of synucleinopathies. To assess the impact of modified α-syn expression prompted by LPS stimulation on the survival of TH positive cells and terminals, the expression of TH was evaluated at 3, 5, and 7 DPI in SN as well as the STR.\u003c/p\u003e \u003cp\u003eIn line with the amplified α-syn expression in the SN at 7 DPI, there was a concurrent decrease in TH expression on the side ipsilateral to the LPS injection at 5 and 7 DPI compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Within the STR, a reduction in TH expression compared to the control group was observed solely on the side ipsilateral to the injection, and solely at 3 DPI, with no discernible changes at 5 or 7 DPI, or comparing with the contralateral side (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Together, these results suggest a rapid degeneration of TH positive (presumably dopaminergic) cells and terminals as a consequence of the reaction triggered by LPS.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe inflammatory process and the consequent aggregation of α-syn are relevant in the development and progression of synucleinopathies; these events can be recapitulated by using intracerebral LPS, which acts via microglial activation and secretion of neurotoxins, generating an inflammatory microenvironment [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In this experiment, we employed intracerebral LPS infusion with the aim of analyzing the expression and early dynamics of α-syn, in the interconnected cerebral nuclei SN and STR once a neuroinflammatory process is triggered, and the influence on the dopaminergic cells and terminals.\u003c/p\u003e \u003cp\u003eOur findings indicate that the inflammatory reaction induced by LPS impacts the expression levels of α-syn and TH in the SN and STR. Additionally, we observed a rapid emergence of small oligomers, ranging from 15 to 60 kDa, exhibiting particular patterns of appearance and expression in the SN and STR. This indicates a cause-and-effect relationship between LPS-induced inflammation and α-syn expression. The general characteristics of the inflammatory reaction following intracerebral LPS infusion have been previously described in murine models [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], along with its ability to increase the accumulation and aggregation of α-syn in the SN [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn contrast to other drugs, such as 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produce a similar outcome within a few hours of damage, the neurodegenerative process triggered by LPS is relatively delayed, since occurs more slowly, resembling the slow progression of synucleinopathy [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], which allows analyzing the presence and abundance of early oligomeric forms of α-syn.\u003c/p\u003e \u003cp\u003eWe observed an increase in the 15 kDa form of α-syn in the STR as early as the third day after LPS administration, but not in the SN. This could indicate the presence of monomers before 3 DPI in the SN, maybe in a physiological abundance sufficient to maintain cellular functions of α-syn, but not enough to be detected by Western blot. In fact, these 15 kDa monomeric forms were detected in the SN up to 7 DPI, supporting the notion of an indetectable quantity of them, and a posterior accumulation in the SN. Furthermore, it is known that the expression of this protein is generally higher in the STR than in the SN, probably due to its affinity for small-sized vesicles, such as presynaptic vesicles [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. To our knowledge, this is the first report of abnormally elevated levels of monomeric α-syn under these conditions as early as 3 days after LPS injection in the nigrostriatal circuit. Nonetheless, the level of monomer expression that we observed in the STR at 3 DPI and in the SN at 7 DPI are not a normal state.\u003c/p\u003e \u003cp\u003eIt has been proposed that the pathogenicity of α-syn is related to the formation of abnormal aggregates, which causes it to lose its normal function, or to gain functions that it did not have before [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Notably, there are no reports of the disruption of cellular functions attributed to α-syn, such as neurotransmitter release or mitochondrial functions [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] under conditions of induced inflammation and neurodegeneration. It will be necessary to delve deeper into this topic, to recognize the perhaps subtle pathological manifestations that we are not showing, in these early stages of α-syn aggregation.\u003c/p\u003e \u003cp\u003eInterestingly, we observed an increase in the expression level of the 60 kDa oligomer (potentially corresponding to a tetrameric α-syn) at 5 DPI in both the SN and the STR. This finding reinforces our hypothesis concerning the instability of the monomer and its transformation to more stable forms, such as the tetramer, in the SN. In addition, it sheds light on the rapid dissemination of these aggregated forms toward the striatum.\u003c/p\u003e \u003cp\u003eRecently, it was suggested that the tetrameric form tends to resist α-syn aggregation as long as it remains with an α-helical configuration. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. However, if these tetramers shift into β-sheets, they can indeed promote α-syn aggregation [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. It is also plausible that α-helix and β-sheet configurations coexist during the initial stages of LPS-induced damage, and depending on the permanency and amplification of the inflammatory process, the balance tips towards self-regulation, or towards dissemination and neurodegeneration. Further research will be imperative to ascertain whether the presence of the tetramer in the α-helix form truly provides neuroprotection and stability. A parallel study noted a significant increase in trimeric α-syn (51 kDa) expression in the SN seven days after LPS stimulation, although other oligomers were not observed [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhile we did not detect significant changes in abundance of 37 or 50 kDa oligomers, another study utilizing intranigral LPS in adult rats identified an increased presence of the 50 kDa form seven days after LPS infusion [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In our study, we administered LPS at a lower concentration (2.5 \u0026micro;g/\u0026micro;L) than Liu et al. (2020) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], trying to mimic a slow onset, as is believed to happen in human synucleinopathy. Although we did not observe a significant increase in 50 kDa oligomers, we obtained evidence of a rise in its expression. Although this increase did not reach the threshold of statistical significance set forth by the study, its presence is indicative of potential neurotoxicity and we consider that it is evidence that the aggregation process is happening [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFinally, we observed a decrease in TH levels at 5 DPI, associated with the presence of various α-syn oligomeric species generated in the SN after LPS infusion. Previous evidence has indicated that α-syn overexpression leads to reduced TH content in the SN [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], with the involvement of Ser40, whose phosphorylation significantly contributes to TH activation and dopamine synthesis [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. This result supports the hypothesis that high levels of α-syn expression are toxic to TH-positive dopaminergic neurons, although our approach does not allow us to determine if there is neurodegeneration or if the detrimental effect is on the expression of the protein.\u003c/p\u003e \u003cp\u003eFew studies have examined the time course of both monomeric and oligomeric α-syn levels within a short timeframe. Our work is the first to detect these aggregates early in an \u003cem\u003ein vivo\u003c/em\u003e model. In other studies, accumulation of α-syn has been observed in the cytoplasm of spared TH-positive neurons four weeks after intrastriatal or intrapallidal LPS administration [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e],25]. In another study, intraperitoneal injection of LPS in aged C57BL/6J mice resulted in an increased abundance of monomeric α-syn one day after LPS administration, although no data were presented regarding oligomeric forms greater than 15 kDa [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Despite differences in the way neuroinflammation is induced, LPS dosage, species, age, and administration method, this result underscores the rapid response of α-syn to brain inflammation, in this case triggered by LPS.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eOur findings conclusively establish that the injection of LPS into the SN swiftly influences α-syn expression, resulting in the formation of oligomers. This finding aligns with the overarching hypothesis that inflammatory processes play a pivotal role in synucleinopathies. Furthermore, the emergence of these oligomeric forms, which are being shown for the first time in this \u003cem\u003ein vivo\u003c/em\u003e animal model, underscores the vulnerability of nigral dopaminergic neurons and their interconnectedness with the STR. This vulnerability becomes evident when potentially toxic forms of α-syn form rapidly following a minimal dose of LPS, impacting both the SN and the STR in a few days, placing a seed that can compromise future brain functioning. Enhancing our comprehension of the cellular and molecular mechanisms driving the onset of α-synucleinopathies will undoubtedly contribute to the future development of therapeutic strategies aimed at mitigating the progression of this pathology.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cul\u003e\n\u003cli\u003eEthical approval was taken from the Research Coordination of the University Campus of Biological and Agricultural Science (CUCBA) of the University of Guadalajara, under the agreement CINV.104/12.\u003c/li\u003e\n\u003cli\u003eAll authors reviewed and authorized the final manuscript and agreed to publish it.\u003c/li\u003e\n\u003cli\u003eAKLL did the experimental work and wrote the first draft under the supervision of SJLP. JLCC helped in data collection and data analysis. SJLP and MEUG critically review the final manuscript. All authors read and approved the final manuscript.\u003c/li\u003e\n\u003cli\u003eThe authors declare no competing interest.\u003c/li\u003e\n\u003cli\u003eThis work received funding from the Consejo Nacional de Humanidades, Ciencia y Tecnolog\u0026iacute;a (CONAHCYT) through scholarship No. 1028543 to AKLL., and from the University of Guadalajara through the P3E-UDG-2022/2023 program to\u003c/li\u003e\n\u003c/ul\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eAKLL did the experimental work and wrote the first draft under the supervision of SJLP. JLCC helped in data collection and data analysis. SJLP and MEUG critically review the final manuscript. All authors read and approved the final manuscript\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eData is provided within the manuscript. 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Nov. 2019.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003edoi: 10.1155/2019/4246350\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"α-syn, oligomers, nigrostriatal circuit, synucleinopathies, tyrosine hydroxylase","lastPublishedDoi":"10.21203/rs.3.rs-4253562/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4253562/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe insoluble tangles of alpha-synuclein (α-syn) protein in the nigrostriatal circuit, characteristic of synucleinopathy, originate from low molecular weight oligomers, whose appearance and dissemination are related to neuroinflammation. These oligomeric forms of α-syn are considered highly cytotoxic but transient, so knowing the timing in which they appear remains a challenge. Therefore, this study aimed to analyze the abundance of oligomeric forms of α-syn and tyrosine hydroxylase (TH) between 1 and 7 days after inducing neuroinflammation with lipopolysaccharide \u003cb\u003e(\u003c/b\u003eLPS).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods and Results\u003c/b\u003e\u003c/p\u003e \u003cp\u003eLPS (2.5 \u0026micro;g/2.5 \u0026micro;L) was stereotaxically injected in the SN of adult male Wistar rats, which were sacrificed 3, 5 and 7 days after this intervention. The brains were processed for semi quantitative Western blot, along with brains from control and sham animals. Our results show an increased expression of α-syn monomer (15 kDa) only 3 days after LPS infusion, and the formation of 50 KDa and 60 kDa α-syn oligomers in the SN and STR between 3 and 7 days after LPS infusion. Furthermore, the presence of these oligomers was accompanied by a decrease in the expression of nigral TH.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThese findings highlight the rapidity with which potentially toxic forms of α-syn appear in the nigrostriatal circuit after a neuroinflammatory challenge, in addition to allowing us to identify specific oligomers and a temporal relation with neurodegeneration of TH-positive cells. Knowledge of the timing and location in which these small oligomers appear is essential to developing therapeutic strategies to prevent its formation.\u003c/p\u003e","manuscriptTitle":"Early expression of monomeric and oligomeric alpha-synuclein and tyrosine hydroxylase following intranigral injection of lipopolysaccharide","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-16 10:36:59","doi":"10.21203/rs.3.rs-4253562/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f9f683d3-47d0-480b-8220-ff09f8399275","owner":[],"postedDate":"April 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-30T14:30:11+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-16 10:36:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4253562","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4253562","identity":"rs-4253562","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}